Phenotypic and genomic analysis of bacteria from war wounds in Dnipro, Ukraine

Abstract Objectives To better understand the source and potential transmission routes of antibiotic-resistant bacteria infecting injured service members in Ukraine. Methods Phenotypic and genomic characterizations were performed on 11 Gram-negative pathogens cultured from war wounds at an intermediate evacuation hospital in Dnipro. Results The analysis revealed both susceptible and extensively drug-resistant strains present in cultures, including high-risk global clones carrying carbapenemases. Conclusions Globally distributed carbapenemase-producing lineages are being acquired early in the medical evacuation process.


Introduction
Before the full-scale invasion by the Russian Federation in February 2022, the prevalence of MDR organisms (MDROs) in Ukraine was estimated at 19.5%. 1 However, this rate varied significantly among species, with a 2022 WHO/ECDC study of Ukraine indicating that carbapenem resistance varied from 4% in Escherichia coli to 53.3% in Klebsiella pneumoniae in 2020 (https://www.ecdc.europa.eu/sites/default/files/documents/Joint-WHO-ECDC-AMR-report-2022.pdf)Recently, a Ukrainian-led international collaboration identified multiple challenges in controlling MDROs and healthcare-associated infections (HAIs), including inadequate surveillance and infection prevention, 2 but the onset of the invasion has greatly exacerbated the problem. 3Notably, these observations reflect previous trends from armed conflicts in Afghanistan, Syria 4 and Iraq, 5 where MDRO infections increased significantly in military and civilian populations.
7][8][9] These have revealed multiple species, especially Acinetobacter baumannii, K. pneumoniae and Pseudomonas aeruginosa carrying an array of antimicrobial resistance (AMR) genes.Critically, these bacteria were obtained from patients near the end of the evacuation process, obscuring their origins and potential transmission routes.In this study, we provide further insights into the epidemiology of these bacteria by performing phenotypic and genomic characterizations of 11 Gram-negative bacteria causing infectious complications of combat-related injuries in Dnipro, an intermediate evacuation hub for war-wounded.

Materials and methods
Three E. coli, three K. pneumoniae and five P. aeruginosa were cultured from 11 injured Ukrainian service members between December 2022 and March 2023 at a hospital in Dnipro (Table 1).Patients were selected based on the severity of trauma and infectious complications from combat injuries with chronic non-healing wounds.Antibiotic susceptibility testing (AST) was performed on site by disc diffusion using EUCAST guidelines (V14.0,2024; www.eucast.org).In addition, all isolates were sent to the Multidrug Resistant Organism Repository and Surveillance Network (MRSN) in the USA, where AST was repeated on a VITEK 2 system, and additional testing performed using broth microdilution or a customized Sensititre plate, as applicable.For cefiderocol testing, broth microdilution was performed in iron-depleted cation-adjusted Mueller-Hinton broth as recommended by the CLSI.All results were interpreted using CLSI guidelines (Table S1, available as Supplementary data at JAC-AMR Online).Finally, WGS was performed on a MiSeq benchtop sequencer, followed by high-resolution genomic analysis, as previously described. 8

Results
The three E. coli were assigned to ST46, ST131 and ST8184 and displayed different susceptibility profiles (Table 1, Table S1).Notably, E. coli MRSN 122312 (ST8184) was susceptible to all antibiotics tested and carried no AMR genes.E. coli MRSN 122283 belonged to Clade A of the globally distributed ST131 10 and was susceptible to 19 of the 29 antibiotics tested.The phenotype correlated with the presence of three AMR genes, including the ESBL bla CTX-M-15 , and point mutations in gyrA (D87N and S83L) and parC (E84V and S80I) that confer fluoroquinolone resistance.E. coli MRSN 122311 was the most concerning and exhibited susceptibility to just 12 of the 29 antibiotics.Notably, the isolate was resistant to both imipenem and meropenem and to cefiderocol (Table S1).Resistance to the carbapenems could be traced to the presence of bla NDM-5 .Similarly, cefiderocol resistance was likely due to the presence of bla NDM-5 and mutations resulting in a non-functional OmpC and a four amino acid substitution (YRIN) at position 333 of penicillin-binding protein 3 (PBP3) in these isolates, as previously described. 11,12The isolate belonged to ST46, a lineage recently implicated in the expansion of E. coli carrying the bla NDM-5 carbapenemase across Europe. 13Similar to those isolates, MRSN 122311 carried bla CTX-M-15 , and a comparison with ST46 genomes available on EnteroBase (enterobase.warwick.ac.uk) revealed a high genetic relatedness (5-51 SNPs) to isolates cultured from patients in Denmark, Germany and the Netherlands (Figure 1a).Limited data are available at the NCBI on these isolates, but the three German isolates and the one Danish isolate were cultured from Ukrainian refugees receiving care in those countries.
The three K. pneumoniae belonged to three different STs, ST23, ST392 and ST395 (Table 1) with distinct antibiotic susceptibility profiles (Table S1).MRSN 122313 was susceptible to 27 of the 28 antibiotics but belonged to the well-known hypervirulence lineage ST23 (KL1), had a Kleborate score 14 of 5, and carried all five virulence markers that best predict a hypervirulent phenotype (iucA, iroB, peg-344, rmpA and rmpA2). 15In contrast, the ST392 (KL27) MRSN 122289 was an ESBL-producer (bla CTX-M-15 ), was susceptible to 19 of the 28 antibiotics tested, had a Kleborate score of 1, and only carried the ybt16 gene (encoding a yersiniabactin siderophore) on a chromosomal ICEKp12.This lineage is globally distributed and has been linked to infections in humans and companion animals. 16Finally, MRSN 122259 was susceptible to just 7 of the 28 antibiotics tested and, similar to E. coli MRSN 122311, was also resistant to cefiderocol and the carbapenems.Carbapenem resistance was conferred by bla NDM-5 , which also contributed to the cefiderocol resistance, and the presence of a mutation leading to a truncated OmpK35, as previously described. 11,17The isolate belonged to the globally emerging carbapenemase-producing lineage ST395 (KL2KL30). 18Similar to other ST395 isolates, it carried the 16S methyltransferase (RMTase) armA, bla NDM-5 and bla CTX-M-15 (Table 1).Notably, the isolate had a Kleborate 14 virulence score of 4 and carried four of the five hypervirulence markers (missing iroB).When compared with other ST395 isolates, it clustered with recent Ukrainian isolates (27-104 SNPs) and historical strains from the USA, the UK and Australia (Figure 1b).
susceptibility and the presence of acquired AMR genes differed significantly.Both ST428 isolates were susceptible to 17 of the 19 antibiotics tested and carried no additional acquired AMR genes.In contrast, both ST773 isolates were susceptible to just two antibiotics, cefiderocol and colistin (Table S1), and carried the same six acquired AMR genes, including the bla NDM-1 carbapenemase and the rmtB4 16S RMTase (Figure 1c).The isolates were most related to one cultured from an injured Ukrainian soldier in 2023 (15-16 SNPs), 8 and more distantly (28-40 SNPs) to strains from the USA and Saudi Arabia obtained between 2017 and 2020 (Figure 1c).Isolate MRSN 122260 (ST1047) shared a similar AST profile with the ST773 isolates, except for a significantly elevated MIC of aztreonam/avibactam (32 mg/L versus 4 mg/L; Table S1).The isolate carried nine acquired AMR genes, including the bla IMP-1 carbapenemase.The isolate was most related (45 SNPs) to an IMP-1-producing strain cultured from the blood of a Ukrainian soldier in 2023 (Figure 1d), 8 and to an NDM-1-producing strain cultured from a respiratory specimen at the Pakistan Institute of Medical Sciences in 2017 (58 SNPs).Four other ST1047 strains collected in the USA between 2019 and 2021 were more distantly related (45 to 110 SNPs) but lacked a carbapenemase.

Discussion
Though only a limited number of strains were included in this study, it provides the first snapshot of bacteria being cultured from war wounds soon after injury.In most evacuation cases, the Dnipro region is an intermediate hub on the way to definitive medical care and rehabilitation further west. 19However, due to the ongoing war, delayed transfers, crowded hospitals and limited infection control resources the risk of infectious complications and HAIs is increased.As demonstrated in this report, both susceptible and MDR strains are being cultured in Dnipro, indicating that MDR strains are already infecting wounds at this point in the evacuation chain.

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Disclaimer
The opinions or assertions contained herein do not necessarily reflect the opinions of the Department of the Army or the Department of Defense.

Table 1 .
Characteristics of the 11 war wound isolates in this study aIn silico-derived multi-locus sequence type.b Month and year isolate was cultured.c